Problems in nucleic acid preparation due to non-nucleic acid components of the nucleic acid source are well-known. For example, the preparation of RNA is complicated by the presence of ribonucleases that degrade RNA. J. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.5 (2d edition, Cold Spring Harbor Laboratory 1989). Furthermore, the preparation of amplifiable RNA is made difficult by the presence of ribonucleoproteins in association with RNA. See R. J. Slater, in Techniques in Molecular Biology, (Macmillan, N.Y. 1983) (J. M. Walker and W. Gaastra, eds.) (pp. 113-120).
Three basic methods are employed for RNA preparation: 1) extraction with phenol; 2) degradation with protease; or 3) disruption and ribonuclease inhibition with strong salts. Phenol is basically a denaturant. While useful, phenol extraction is time consuming and creates a serious waste disposal problem. Use of protease requires the addition of a detergent (e.g., SDS); detergents must be removed for the recovered RNA to be useful in subsequent assays. Finally, the use of salts alone does not result in the purification of RNA that is free of protein; current protocols require the use of salts in conjunction with phenol (P. Chomczynski and N. Sacchi, Anal. Biochem. 162:156 (1987)) or employ a centrifugation step to remove the protein (R. J. Slater, supra).
Where purification of RNA is for the purpose of producing template for amplification, it is important to consider the source (i.e., bone marrow, spinal fluid, urine, feces, etc.) and potential polymerase inhibitors that are constituents in such sources. One class of constituents known to inhibit nucleic acid associated enzymes are the "hemes" which include hemin and hematin. Hemin has been reported to inhibit virion-associated reverse transcriptase (RTase) of murine leukemia virus (MuLV) (Tsutsui and Mueller, BBRC 149:628-634, 1987), DNA ligase (Scher et al., Cancer Res. 48:6278-6284, 1988), cytoplasmic DNA polymerase (Byrnes et al., Biochem. 14:796-799, 1975), Taq polymerase (PCR Technology, H. A. Erlich (ed.) Stockton Press (1989) p. 33), and other enzymatic systems that utilize ATP as a cofactor such as the hemin-controlled protein kinase that affects protein synthesis (Hronis and Traugh, J. Biol. Chem. 261:6234-6238, 1986), the ATP-dependent ubiquitin-dependent protease pathway (Hershko et al., Proc. Natl. Acad. Sci. USA 81:1619-1623, 1984), and the ATP-dependent ubiquitin-independent protease pathway (Waxman et al., J. Biol. Chem. 260:11994-12000, 1985).
Freshly-made hemin solution inhibited MuLV RTase activity by 50% at a hemin concentration of 10 .mu.M. Aged hemin solutions (5 days at room temperature) inhibited MuLV RTase by 50% at 0.1 .mu.M concentration. Addition of 4-fold excess MuLV RTase caused an increase of enzyme activity in the presence of hemin while addition of excess template did not. Addition of a heme-binding protein from rabbit serum (Tsutsui and Mueller, J. Biol. Chem. 257:3925-3931, 1982) completely restored enzyme activity. This suggests that hemin is a reversible inhibitor of MuLV RTase and that its interaction with the enzyme is noncovalent in nature. Hemin does not inhibit the activity of reverse transcriptase purified from arian myeloblastosis virus.
Experiments with DNA ligase indicate that hemin at 4 .mu.M or less does not affect DNA ligase activity or DNA substrate integrity. Scher et al., supra. Pre-incubations of DNA ligase with hemin led to half-maximal inhibition of DNA ligase at hemin concentrations of 25-100 .mu.M (depending on the source of the DNA ligase). NAD-dependent DNA ligase from E. coli was not inhibited by hemin at concentrations up to 150 .mu.M. The inhibition of T4 DNA ligase activity and DNA ligase from mouse erythroleukemia (MEL) cells was not reversible by dilution, dialysis, or sucrose gradient centrifugation of cell-free extracts. Incubation of DNA ligase from MEL cells with hemoglobin was not inhibitory.
Binding assays demonstrate that hemin prevents association and causes dissociation of the DNA-cytoplasmic DNA polymerase complex. Hemin at a concentration of 12 .mu.M or higher completely inhibits the formation of DNA-enzyme complex. Byrnes et al., supra. This report also shows that hemin inhibition of DNA synthesis is competitive with respect to template and noncompetitive with respect to substrate. Furthermore, inhibition could be reversed by either: 1) addition of globin to the polymerase-containing reaction mixture prior to the addition of hemin to the reaction mixture; or 2) addition of globin to hemin followed by addition of this mixture to the polymerase-containing reaction mixture. Inhibition could not be reversed by the addition of globin after introduction of hemin to the polymerase-containing reaction mixture.
Experiments with purified hematin and related compounds have shown that they are potent inhibitors of Taq polymerase. Taq polymerase is used in the amplification procedure described by K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202. This amplification procedure is a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle;" there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to by the inventors as the "Polymerase Chain Reaction" (hereinafter PCR). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified".
Hematin is inhibitory to PCR at a final concentration of 0.8 .mu.M or higher (PCR Technology, H. A. Erlich (ed.) Stockton Press (1989) p. 33). "Protoporphyrin" is inhibitory at 20 .mu.M. Non-heme blood components such as globin, Fe++ and Fe.sup.+++ ions also inhibit PCR (Walsh et al.).
Where the inhibitor is a competitive inhibitor, one approach is to add more reagent and "swamp" the inhibition. This has been attempted in the case of PCR inhibition. Walsh et al. Proc. Int'l. Symp. Forensic Aspects DNA Anal., (Jun. 19-23, 1989), have shown that, while hematin inhibition cannot be overcome by additional quantities of template DNA, it can be overcome by additional quantities of Taq polymerase or primer.
This swamping approach has a serious disadvantage: additional quantities of reagents may cause spurious results. Indeed, in the case of PCR it is known that additional quantities of Taq polymerase or primers can result in nonspecific amplification products. S. Paabo et al., Nucleic Acid Res. 16:9775 (1988). These nonspecific products are believed to be due to weak priming sites.
The conventional method for the preparation of amplifiable nucleic acid from whole blood involves isolation of lymphocytes by density gradient centrifugation. This typically involves the isolation of T4 enriched white blood cells by centrifugation through a Ficoll gradient. See e.g., Longley and Stewart, J. Immunol. Methods, 121:33-38, 1989. The red blood cells and granulocytes pellet in this system. The lymphocyte-enriched white blood cells are recovered from the gradient interface. To remove Ficoll, which inhibits Taq polymerase, the cells are usually washed one or more times by centrifuging and removing the supernatant. Although this procedure yields lymphocytes free of red blood cells and most of the platelets, there are a number of disadvantages to this procedure, including: (1) relatively large, freshly drawn blood samples must be layered over Ficoll carefully so that the interface is undisturbed; (2) lymphocytes must be collected (after centrifugation) by removing the opaque band of cells located at the gradient interface; and (3) the collected lymphocytes must be washed free of Ficoll. The careful layering of blood is a slow and somewhat artful step. The collection of the cells at the gradient interface demands that: i) enough blood be used initially such that the cells can be seen with the naked eye; ii) the cells be captured in a pipette (a cumbersome and low-yield technique); and iii) amplification be carried out in a different reaction vessel from that used to layer the blood. Finally, the approach utilizes a polymerase inhibitor (i.e., Ficoll) in large amounts that is not easily removed except by centrifugation. These drawbacks have seriously hindered the application of amplification techniques to large-scale clinical diagnostics.